Composite

Part:BBa_K4307033

Designed by: Chi Zhang   Group: iGEM22_Tsinghua   (2022-09-30)


J32100-AffiPmrB-PmrA-PmrC-T7 core-T3 sigma-pT3-EGFP

This part is improved from our AffiPmrB/A/C part (BBa_K4307028). The fluorescence signal output by the engineered bacteria with AffiPmrB/A/C was very weak. Since our goal was to observe the fluorescence signal with naked eyes, we introduced the T7-T3 cascade amplifier into our Affi-Pmr system to amplify the output fluorescence signal output. T7-core-T3-sigma system is developed from T7 and T3 bacterial phage, the core enzyme of T7 RNA polymerase could interact with the T3 sigma transcription factor, precisely promote the expression of pT3 promoter. We put the T3 sigma under PmrC, thus the signal produced by AffiPmrB/A/C would be amplified and showed as the EGFP fluorescence intensity.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal suffix found in sequence at 2035
    Illegal XbaI site found at 54
    Illegal SpeI site found at 4536
    Illegal SpeI site found at 4749
    Illegal PstI site found at 395
    Illegal PstI site found at 683
    Illegal PstI site found at 1115
    Illegal PstI site found at 4550
    Illegal PstI site found at 4735
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 6736
    Illegal NheI site found at 6759
    Illegal SpeI site found at 2036
    Illegal SpeI site found at 4536
    Illegal SpeI site found at 4749
    Illegal PstI site found at 395
    Illegal PstI site found at 683
    Illegal PstI site found at 1115
    Illegal PstI site found at 2050
    Illegal PstI site found at 4550
    Illegal PstI site found at 4735
    Illegal NotI site found at 2043
    Illegal NotI site found at 4543
    Illegal NotI site found at 4740
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 946
    Illegal BamHI site found at 3316
    Illegal BamHI site found at 4885
    Illegal XhoI site found at 7636
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal suffix found in sequence at 2036
    Illegal XbaI site found at 54
    Illegal SpeI site found at 4536
    Illegal SpeI site found at 4749
    Illegal PstI site found at 395
    Illegal PstI site found at 683
    Illegal PstI site found at 1115
    Illegal PstI site found at 4550
    Illegal PstI site found at 4735
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 54
    Illegal SpeI site found at 2036
    Illegal SpeI site found at 4536
    Illegal SpeI site found at 4749
    Illegal PstI site found at 395
    Illegal PstI site found at 683
    Illegal PstI site found at 1115
    Illegal PstI site found at 2050
    Illegal PstI site found at 4550
    Illegal PstI site found at 4735
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 6881


Characterization

The following figure demonstrates our successful construction.


bba-k4307028-gel.png

               Figure 1:  The construction results of J32100-AffiPmrB-PmrA-PmrC-T7 core-T3 sigma-pT3-EGFP.


The engineered DH5α was incubated with different concentration of IgG. After 10h incubation, the EGFP fluorescence intensity of the DH5α was detected by microplate reader:

bba-k4307028-gel.png

               Figure 2:  We can see that the EGFP fluorescence/OD600 is much higher than AffiPmrB/A/C system (both induced and uninduced), while the bacteria induced by 200μM IgG has the highest EGFP fluorescence intensity, which is about 40% higher than the non-induced bacteria.


Conclusion

The T7-T3 am

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